Showing papers on "Cyclase published in 1989"
TL;DR: A novel neuropeptide which stimulates adenylate cyclase in rat anterior pituitary cell cultures was isolated from ovine hypothalamic tissues and increased release of growth hormone, prolactin, corticotropin and luteinizing hormone from superfused rat pituitaries at as small a dose as 10(-10)M) or 10(-9)M (LH).
1,815 citations
TL;DR: A panel of antibodies to synthetic decapeptides corresponding to the C termini of guanine nucleotide-binding regulatory protein (G protein) alpha subunits has been generated in rabbits and identifies Gi2 as the dominant mediator of cyclase inhibition in this pathway.
Abstract: A panel of antibodies to synthetic decapeptides corresponding to the C termini of guanine nucleotide-binding regulatory protein (G protein) alpha subunits has been generated in rabbits. The specificity of each antibody was assessed by ELISA for peptide binding and by immunoblotting for binding to defined, recombinant G alpha subunits expressed in Escherichia coli. Immunoblotting of human platelet membranes with these antibodies identified a variety of endogenous G proteins including Gs (stimulatory), Gi2 (inhibitory), Gi3, and Gx(z) (unknown function). Pretreatment of platelet membranes with C-terminal antibodies reactive with Gi2, but not with antibodies to Gi3 or Gx(z), blocked alpha 2-adrenergic inhibition of adenylyl cyclase. This identifies Gi2 as the dominant mediator of cyclase inhibition in this pathway. This approach may provide a general means of identifying relevant functional interactions of G proteins with receptors and effectors in situ.
336 citations
TL;DR: It is shown that the protein kinase-like domain functions as a regulatory element and that the second domain possesses catalytic activity in the plasma membrane form of guanylate cyclase.
Abstract: A plasma membrane form of guanylate cyclase is a cell surface receptor for atrial natriuretic peptide (ANP). In response to ANP binding, the receptor-enzyme produces increased amounts of the second messenger, guanosine 3',5'-monophosphate. Maximal activation of the cyclase requires the presence of adenosine 5'-triphosphate (ATP) or nonhydrolyzable ATP analogs. The intracellular region of the receptor contains at least two domains with homology to other proteins, one possessing sequence similarity to protein kinase catalytic domains, the other to regions of unknown function in a cytoplasmic form of guanylate cyclase and in adenylate cyclase. It is now shown that the protein kinase-like domain functions as a regulatory element and that the second domain possesses catalytic activity. When the kinase-like domain was removed by deletion mutagenesis, the resulting ANP receptor retained guanylate cyclase activity, but this activity was independent of ANP and its stimulation by ATP was markedly reduced. A model for signal transduction is suggested in which binding of ANP to the extracellular domain of its receptor initiates a conformational change in the protein kinase-like domain, resulting in derepression of guanylate cyclase activity.
320 citations
TL;DR: Increased endothelial cell cyclic adenine nucleotide activity was an important determinant of endothelial barrier function in vitro, and influences on cytoskeletal assembly may be involved in this process.
Abstract: Cyclic adenosine monophosphate (AMP) has numerous important effects on cell structure and function, but its role in endothelial cells is unclear. Since cyclic AMP has been shown to affect transmembrane transport, cell growth and morphology, cellular adhesion, and cytoskeletal organization, it may be an important determinant of endotelial barrier properties. To test this we exposed bovine pulmonary artery endothelial cell monolayers to substances known to increase cyclic AMP and measured their effect on endothelial permeability to albumin and endothelial cell cyclic AMP concentrations. Cholera toxin (CT), a stimulant of the guanine nucleotide binding subunit of adenylate cyclase, led to a concentration-dependent 2-6-told increase in cyclic AMP which was associated with a 3–10-fold reduction in albumin transfer across endothelial monolayers. The effect was not specific to albumin as similar barrier-enhancing effects were also noted with an unrelated macromolecule, fluorescein isothiocyanate (FITC)-dextran (MW 70,000). Barrier enhancement with cyclic AMP elevation was also observed with forskolin, a stimulant of the catalytic subunit of adenylate cyclase. The temporal pattern of barrier enhancement seen with these agents paralleled their effects on increasing cyclic AMP, and the barrier enhancement could be reproduced by incubation with either dibutyryl cyclic AMP or Sp-cAMPS, cyclic AMP-dependent protein kinase agonists. Furthermore, the forsko-lin effect on barrier enhancement was partially reversed with Rp-cAMPS, an antagonist of cyclic AMP-dependent protein kinase. Since endothelial actin polymerization may be an important determinant of endothelial barrier function, we sought to determine whether the cyclic AMP-induced effects were associated with increases in the polymerized actin pool (F-actin). Both cholera toxin and forskolin led to apparent endothelial cell spreading and quantitative increases in endothelial cell F-actin fluorescence. In conclusion, increased endothelial cell cyclic adenine nucleotide activity was an important determinant of endothelial barrier function in vitro. The barrier enhancement was associated with increased endothelial apposition and increases in F-actin, suggesting that influences on cytoskeletal assembly may be involved in this process.
257 citations
TL;DR: Sucrose and other saccharides, which produce an appealing taste in rats, were found to significantly stimulate the activity of adenylate cyclase in membranes derived from the anterior-dorsal region of rat tongue, consistent with a sweet-taste transduction mechanism involving specific receptors, a guanine-nucleotide-binding protein and the cyclic AMP-generating enzyme.
Abstract: Sucrose and other saccharides, which produce an appealing taste in rats, were found to significantly stimulate the activity of adenylate cyclase in membranes derived from the anterior-dorsal region of rat tongue. In control membranes derived from either tongue muscle or tongue non-sensory epithelium, the effect of sugars on adenylate cyclase activity was either much smaller or absent. Sucrose enhanced adenylate cyclase activity in a dose-related manner, and this activation was dependent on the presence of guanine nucleotides, suggesting the involvement of a GTP-binding protein ('G-protein'). The activation of adenylate cyclase by various mono- and di-saccharides correlated with their electrophysiological potency. Among non-sugar sweeteners, sodium saccharin activated the enzyme, whereas aspartame and neohesperidin dihydrochalcone did not, in correlation with their sweet-taste effectiveness in the rat. Sucrose activation of the enzyme was partly inhibited by Cu2+ and Zn2+, in agreement with their effect on electrophysiological sweet-taste responses. Our results are consistent with a sweet-taste transduction mechanism involving specific receptors, a guanine-nucleotide-binding protein and the cyclic AMP-generating enzyme adenylate cyclase.
179 citations
Journal Article•
TL;DR: A cyclic AMP independent transduction pathway for DA-1 receptor mediated through a guanine nucleotide regulatory protein associated phospholipase-C is indicated.
Abstract: Dopamine-1 (DA-1) receptors have been found in renal tubular membranes which stimulate both adenylate cyclase and phospholipase-C activity. In renal cortical plasma membrane preparations the DA-1 agonist SKF 82526, forskolin and NaF stimulated adenylate cyclase activity. 2',5'-dideoxyadenosine inhibited basal and DA-1 agonist stimulated adenylate cyclase activity. Forskolin, NaF, dibutyryl-cyclic AMP and 2',5'-dideoxyadenosine had no effect on basal or DA-1 agonist stimulated phospholipase-C activity in these membranes. These studies indicate that DA-1 agonist stimulates adenylate cyclase and phospholipase-C activities independently. Phospholipase-C activity was also increased by the nonhydrolyzable GTP analog, guanosine-5'-O-(3-thiophosphate). When DA-1 agonist and guanosine-5'-O-(3-thiophosphate) were added together there was a slight but significant increase in phospholipase-C activity. This increase was inhibited in the presence of guanosine-5'-O-(2-thiodiphosphate). DA-1 stimulated phospholipase-C activity was found to be insensitive to both cholera and pertussis toxins. The present studies indicate a cyclic AMP independent transduction pathway for DA-1 receptor mediated through a guanine nucleotide regulatory protein associated phospholipase-C.
171 citations
TL;DR: A membrane form of guanylate cyclase represents a new model for cell surface receptors, although such a model was once envisioned for adenylate cyclase.
137 citations
Journal Article•
TL;DR: Northern analysis demonstrated that mRNAs for GS alpha and all three isoforms of Gi alpha are present in adipocytes but that there are no changes in the levels of any of these transcripts following desensitization, suggesting that down-regulation of A1ARs and an additional loss of A 1AR agonist high affinity sites does not occur by altering the levelsof their respective transcripts.
Abstract: The A1-adenosine receptor (A1AR) adenylate cyclase system in rat adipocytes undergoes heterologous desensitization following chronic in vivo exposure to an A1AR agonist (+)-N6-(R-phenylisopropyl)adenosine [J. Biol. Chem. 262:841-847 (1987)]. This desensitization involves an absolute increase in adenylate cyclase activity and a refractoriness to receptor ligands that are inhibitory to adenylate cyclase. In this study, receptor changes were characterized using an A1AR antagonist radioligand, [3H]8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl] oxy]phenyl]-1,3-dipropyl xanthine. Saturation binding studies demonstrated a 47% decrease in total A1AR density without a change in KD. Agonist competition studies revealed a decreased percentage of receptors, from 55% to 35%, in the high affinity state following desensitization. An increase in GS alpha of 49% was found by Western blotting using specific GS alpha antibodies. Further, an antibody that recognizes Gi alpha 1 adn Gi alpha 2 was used to quantitate these subtypes of Gi alpha and both were decreased by 59% following desensitization. However, when an antibody that recognizes Gi alpha 3 was used, no change in Gi alpha 3 was found, demonstrating, in this case, differential regulation of Gi alpha subtypes. The mechanisms responsible for changes in GS alpha and Gi alpha were studied by measuring the levels of their mRNAs from normal and desensitized adipocytes. Using either labeled cDNAs (Gi alpha 2, Gi alpha 3) or oligonucleotides (GS alpha, Gi alpha 1), Northern analysis demonstrated that mRNAs for GS alpha and all three isoforms of Gi alpha are present in adipocytes but that there are no changes in the levels of any of these transcripts following desensitization. These data suggest that desensitization of the A1AR-adenylate cyclase system involves a down-regulation of A1ARs and an additional loss of A1AR agonist high affinity sites. Further, an increase in GS alpha, a decrease in Gi alpha 1 and Gi alpha 2, and no change in Gi alpha 3 were found. The regulation of GS alpha and the subtypes of Gi alpha in this system does not occur by altering the levels of their respective transcripts.
124 citations
TL;DR: Electroelution of the 216-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis yields a preparation with both AC enzyme and toxin activities, indicating that this protein represents the AC holotoxin molecule.
124 citations
Journal Article•
TL;DR: The cation and structural requirements of the intracellular inhibitory "P" site of adenylate cyclase were investigated in human platelet membranes, bovine sperm particles, and detergent-solubilized and purified preparations from rat and bovines brain, suggesting a strict requirement for an intact adenine moiety and a beta-glycosidic linkage for the ribosyl moiety.
Abstract: The cation and structural requirements of the intracellular inhibitory "P" site of adenylate cyclase were investigated in human platelet membranes, bovine sperm particles, and detergent-solubilized and purified preparations from rat and bovine brain. Sensitivity of adenylate cyclase to P site-mediated inhibition was enhanced by reversible and irreversible activators of the enzyme. The most effective sensitization of the platelet and brain adenylate cyclases was observed with Mn2+ and upon proteolysis with inhibin in the presence of guanosine 5'-O-(3-thiotriphosphate). These resulted in IC50 values for (2',5'dideoxy-adenosine (2',5'-dd-Ado) and 2'-deoxy-3'-AMP of approximately 1-2 microM. The data were consistent with the ideas that P site-mediated inhibition of adenylate cyclase is dependent on divalent cation and is a function of enzyme activity. A number of nucleosides and nucleotides were synthesized and used to define structural requirements for P site-mediated inhibition of a detergent-solubilized adenylate cyclase from rat brain. The data suggest a strict requirement for an intact adenine moiety and a beta-glycosidic linkage for the ribosyl moiety. 2'-Deoxy-and especially 2',5'-dideoxy-ribosyl moieties enhanced sensitivity and a strong preference for phosphate at the 3'-position was exhibited. Substitutions at the 5'-ribose position impaired sensitivity. The order of potency and IC50 values of the more potent adenosine analogs were 2',5'-dideoxy-3'-AMP (congruent to 0.1 microM) greater than 2'-deoxy-3'-AMP (congruent to 1 microM) greater than 2',5'-dd-Ado (congruent to 3 microM) greater than 3'-AMP (congruent to 9 microM) greater than 2'-deoxy-adenosine (congruent to 15 microM) greater than adenosine (congruent to 80 microM). Large substitutions at the 3'-ribose position were tolerated, e.g., dApdN di- and dAp(dN)4 penta-nucleotides and succinyl- and p-fluoro-sulfonyl-benzoyl- moieties. The purified adenylate cyclase from bovine brain was inhibited by P site agonists with IC50 values of 34 and 45 microM for 2'-deoxy-3'-AMP and 2',5'-dd-Ado, respectively. The data imply, first, that the locus of the P site is the catalytic subunit of adenylate cyclase and, second, that the increased sensitivity observed with Mn2+ is due to an effect of the cation on the catalytic subunit. In contrast with adenylate cyclases from other mammalian tissues, the enzyme from bovine sperm exhibited only weak sensitivity to P site agonists; 2'-deoxy-3'-AMP congruent to 2',5'-dd-Ado greater than adenosine, each with IC50 values greater than 1000 microM.(ABSTRACT TRUNCATED AT 400 WORDS)
118 citations
TL;DR: A strong positive correlation between the magnitude of adenylate cyclase stimulation and the summated electrical response of the olfactory epithelium (electro-olfactogram) evoked by individual odorants is reported.
Abstract: An odor-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] is thought to mediate olfactory transduction in vertebrates. However, it is not known whether the adenylate cyclase serves this function for all odorants or for only certain classes of odorants. To investigate this question, we have compared the abilities of 35 odorants to stimulate the adenylate cyclase and to elicit an electrophysiological response. We report a strong positive correlation between the magnitude of adenylate cyclase stimulation and the summated electrical response of the olfactory epithelium (electro-olfactogram) evoked by individual odorants. We also show that the adenylate cyclase stimulator forskolin equally attenuates the electro-olfactogram response for all odorants tested. These data provide evidence that the adenylate cyclase mediates transduction for a wide variety of odorants.
TL;DR: It is found that K‐opiate receptors are negatively coupled to adenylate cyclase, and the pattern of heterologous desensitization suggests that chronic exposure to K opiates leads to alterations in postreceptor regulatory components, possibly GTP‐binding proteins.
Abstract: The nature of the opiate modulation of adenylate cyclase following acute and chronic agonist exposure has been investigated in rat spinal cord. Using membranes of both adult rat spinal cord and spinal cord-dorsal root ganglion cocultures, we found that kappa-opiate receptors are negatively coupled to adenylate cyclase. The kappa-opiate agonists (e.g., U50488) inhibit significantly and dose-dependently the basal and the forskolin-stimulated cyclase activities, whereas mu and delta agonists are ineffective. The regulatory action is stereospecific and requires the presence of GTP. EGTA treatment of the plasma membranes abolished the effect of kappa-opiate agonists on the basal cyclase activity, and this inhibitory effect could not be restored by subsequent addition of Ca2+. The EGTA treatment did not affect the kappa agonist inhibition of the forskolin-stimulated cyclase. The results also show that following chronic exposure of cultured cells to etorphine or U50488, there is a loss of kappa agonist inhibition of the cyclase. Moreover, this desensitization process appears to be heterologous, because alpha 2-adrenergic agonists (e.g., clonidine or norepinephrine) and the muscarinic agonist (carbachol) exhibited significantly lower potency for inhibiting cyclase activity when compared to untreated cultures. This pattern of heterologous desensitization suggests that chronic exposure to kappa opiates leads to alterations in postreceptor regulatory components, possibly GTP-binding proteins.
TL;DR: The data indicate that prolonged treatment of rat heart muscle cells with noradrenaline leads to an increase in the level of alpha-subunits of Gi-proteins, which is responsible for the observed heterologous desensitization of adenylate cyclase stimulation.
TL;DR: The results substantiated, at the molecular level, the previous genetic and biochemical studies according to which the N‐terminal tryptic fragment of secreted B.pertussis adenylate cyclase harbours the catalytic site, whereas the C-terminaltryptic fragment corresponds to the main CaM‐binding domain of the enzyme.
Abstract: In order to identify molecular features of the calmodulin (CaM) activated adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the recombinant protein are very similar to that of adenylate cyclase purified from B.pertussis culture supernatants, i.e. a specific activity greater than 2000 mumol/min mg of protein at 30 degrees C and pH 8, a KmATP of 0.6 mM and a Kd for its activator, CaM, of 0.2 nM. Proteolysis with trypsin in the presence of CaM converted the recombinant protein to a 43 kd protein with no loss of activity; the latter corresponds to the secreted form of B.pertussis adenylate cyclase. Site-directed mutagenesis of residue Trp-242 in the recombinant protein yielded mutants expressing full catalytic activity but having altered affinity for CaM. Thus, substitution of an aspartic acid residue for Trp-242 reduced the affinity of adenylate cyclase for CaM greater than 1000-fold. Substitution of a Gln residue for Lys-58 or Lys-65 yielded mutants with a drastically reduced catalytic activity (approximately 0.1% of that of wild-type protein) but with little alteration of CaM-binding. These results substantiated, at the molecular level, our previous genetic and biochemical studies according to which the N-terminal tryptic fragment of secreted B.pertussis adenylate cyclase (residues 1-235/237) harbours the catalytic site, whereas the C-terminal tryptic fragment (residues 235/237-399) corresponds to the main CaM-binding domain of the enzyme.
TL;DR: A new generation of vectors for the hyperexpression of lethal gene products such as adenylate cyclase in Escherichia coli is described, based on the lambda PL promoter and lambda cII ribosome binding site described by Shimatake and Rosenberg.
Abstract: We describe the construction of a new generation of vectors (pRE) for the hyperexpression of lethal gene products such as adenylate cyclase in Escherichia coli. The pRE vectors are based on the lambda PL promoter and lambda cII ribosome binding site described by Shimatake and Rosenberg (Nature, 292, 128-132, 1981). They have a unique NdeI restriction endonuclease site 3' of the lambda cII ribosome binding site that includes the ATG initiation codon, multilinker cloning sites 3' to the NdeI site, and two lambda transcription terminators 5' and 3' of the lambda PL promoter to eliminate nonspecific transcription and reduce leaky PL transcription, respectively. For hyperexpression of adenylate cyclase, tight control of transcription was necessary since elevation of cAMP levels above the physiological range is lethal to E. coli. Lethality associated with the overproduction of adenylate cyclase was shown to be mediated through the cAMP receptor protein. We used this expression system to overproduce adenylate cyclase 7500 fold, corresponding to 30% of the total cellular protein. Under these conditions the enzyme precipitated with significant loss of activity. Reducing the rate and amount of adenylate cyclase expression to 16% of the total cell protein produced one fourth of the enzyme in a soluble form with high specific activity. The soluble adenylate cyclase was purified to near homogeneity.
TL;DR: It is concluded that alpha 2-adrenergic receptors on human erythroleukemia cells couple to mobilization of intracellular Ca2+ via a (pertussis toxin-sensitive) G protein-mediated mechanism that is independent of inhibition of adenylate cyclase.
TL;DR: Results demonstrate that both fragments of the 43-kDa form of adenylate cyclase are essential for a high level of enzymatic activity.
TL;DR: Results suggest that the PGE receptor is coupled to two signal transduction systems leading to inhibition of cAMP accumulation via Gi and to production of inositol phosphates via a pertussis toxin-insensitive G-protein, both of which may modulate catecholamine release from bovine chromaffin cells.
TL;DR: The data suggest that gamma-aminobutyric acid (GABAA) receptor function in brain can be regulated by cAMP-dependent phosphorylation, similar to the time course for cAMP accumulation.
Abstract: The effects of the cyclic nucleotide cAMP on gamma-aminobutyric acid-gated chloride channel function were investigated. The membrane-permeant cAMP analog N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate inhibited muscimol-induced 36Cl- uptake into rat cerebral cortical synaptoneurosomes in a concentration-dependent manner (IC50 = 1.3 mM). The inhibition was due to a decrease in the maximal effect of muscimol, with no change in potency. Similar effects were observed with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 8-bromoadenosine 3',5'-cyclic monophosphate, and the phosphodiesterase inhibitor isobutylmethylxanthine. The effect of endogenous cAMP accumulation on the gamma-aminobutyric acid-gated Cl- channel was studied with forskolin, an activator of adenylate cyclase. Under identical conditions, in the intact synaptoneurosomes, forskolin inhibited muscimol-induced 36Cl- uptake and generated cAMP with similar potencies (IC50 = 14.3 microM; EC50 = 6.2 microM, respectively). Surprisingly, 1,9-dideoxyforskolin, which does not activate adenylate cyclase, also inhibited the muscimol response, suggesting that forskolin and its lipophilic derivatives may interact with the Cl- channel directly. Indeed, forskolin inhibition of muscimol-induced 36Cl- uptake was extremely rapid (within 5 sec), preceding the accumulation of sufficient levels of cAMP. After 5 min, a slower phase of inhibition was seen, similar to the time course for cAMP accumulation. The data suggest that gamma-aminobutyric acid (GABAA) receptor function in brain can be regulated by cAMP-dependent phosphorylation.
TL;DR: Results suggest that Schizosaccharomyces pombe cyclase may have lost the ability to interact with RAS proteins by the loss of a regulatory site.
Abstract: A Schizosaccharomyces pombe gene encoding adenylate cyclase has been cloned by cross-hybridization with the Saccharomyces cerevisiae adenylate cyclase gene. The protein encoded consists of 1692 amino acids and has adenylate cyclase activity that cannot be activated by the Sa. cerevisiae RAS2 protein. Sc. pombe cyclase has a high degree of homology (approximately 60%) with the catalytic domain of Sa. cerevisiae cyclase precisely mapped by a gene-deletion analysis. A 25-40% identity is observed throughout the middle segments of approximately 1000 residues of both cyclases, large parts of which are composed of repetitions of a 23-amino acid motif similar to those found in human glycoproteins, Drosophila chaoptin, and Toll gene product. However, a segment corresponding to the NH2-terminal 620 residues of Sa. cerevisiae cyclase appears lost from Sc. pombe cyclase, and the COOH-terminal 140 residues are not well conserved between the two yeast species. Deletions involving the COOH-terminal residues of Sa. cerevisiae cyclase cause loss of activation by the RAS2 protein. These results suggest that Sc. pombe cyclase may have lost the ability to interact with RAS proteins by the loss of a regulatory site.
TL;DR: Bordetella pertussis produces an adenylate cyclase which is a toxin that penetrates eukaryotic cells and, upon activation by host calmodulin, generates high levels of intracellular cAMP; as a result bactericidal functions of immune effector cells are considerably impaired.
TL;DR: The purification and characterization of the toxic form of the enzyme, which penetrates eukaryotic cells and generates high levels of intracellular cAMP is reported, which was purified from an extract of B.pertussis strain carrying a recombinant plasmid which over‐produced both enzymatic and toxic activities of the enzymes.
Abstract: Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase (AC) which is an essential virulence factor in mammalian pertussis. Here we report the purification and characterization of the toxic form of the enzyme, which penetrates eukaryotic cells and generates high levels of intracellular cAMP. This form was purified from an extract of B.pertussis strain carrying a recombinant plasmid which over-produced both enzymatic and toxic activities of the enzyme. Western blot analysis of the extract using anti-B.pertussis AC antibodies detected only one protein of 200 kd. However, gel filtration of the extract resolved two peaks of enzymatic activity. The first peak of aggregated material contained greater than 70% of the total enzymatic activity, and the second peak contained the majority of the toxic activity. Purification of the enzyme from both peaks yielded proteins of 200 kd, with similar biochemical and immunological properties. Yet only the enzyme purified from the second peak could penetrate human lymphocyte and catalyse the formation of intracellular cAMP. B.pertussis AC gene expressed in Escherichia coli produced a calmodulin-dependent enzyme of 200 kd, which lacked lymphocyte penetration capacity. It is proposed that a post-translational modification that occurs in B.pertussis but not in E.coli confers upon the 200 kd protein of B.pertussis AC the toxic properties.
TL;DR: The present results suggest that lithium ex vivo decreases neurotransmitter activation of the cortical beta-adrenergic adenylate cyclase by influencing the mechanisms by which receptor agonists enhance the GTP stimulation of the adenyate cyclases.
Journal Article•
TL;DR: The results of these studies suggest that chronic antidepressant administration promoted increased coupling between Gs and catalytic unit of adenylate cyclase, suggesting the molecular locus of antidepressant action may reside at the stimulatory GTP-binding protein, Gs.
Abstract: In an attempt to resolve a unified postreceptor mechanism of action for antidepressant therapy, rats were treated with amitriptyline, desipramine or iprindole. Chronic treatment with these antidepressant drugs increased guanylylimidodiphosphate-[Gpp(NH)p-], NaF-, or forskolin-activated adenylate cyclase in synaptic membranes prepared from cerebral cortexes of treated rats. Gpp(NH)p-dependent inhibition of adenylate cyclase was unaffected. Maximal binding of the photoaffinity GTP analog azidoanilido-GTP (AAGTP) to the adenylate cyclase stimulatory (Gs alpha) and inhibitory (Gi alpha) G proteins was unaffected by antidepressant treatment. The chemical elimination of Gs (low pH treatment) eliminated all differences between control and antidepressant-treated groups. Further, nonneural tissues from rats receiving chronic antidepressants showed no changes in adenylate cyclase activity or AAGTP binding. The results of these studies suggest that chronic antidepressant administration promoted increased coupling between Gs and catalytic unit of adenylate cyclase. Thus, the molecular locus of antidepressant action may reside at the stimulatory GTP-binding protein, Gs.
TL;DR: It is suggested that protein kinase C can elicit the phosphorylation and functional inactivation of α‐Gi in intact hepatocytes as pertussis toxin only causes the ADP‐ribosylation of the holomeric form of Gi, and it may be that phosphorylated leaves α‐ Gi in its holomersic state.
TL;DR: Mutants of Vibrio fischeri MJ-1 apparently deficient in adenylate cyclase (cya-like) or cyclic AMP receptor protein (crp- like) were isolated and characterized and addition of cyclicAMP restored wild-type levels of luminescence and luciferase in the cya- like mutant but not in the crp-like mutant.
Abstract: Mutants of Vibrio fischeri MJ-1 (wild type) apparently deficient in adenylate cyclase (cya-like) or cyclic AMP receptor protein (crp-like) were isolated and characterized. Compared with MJ-1, the mutants produced low levels of luminescence and luciferase. Addition of cyclic AMP restored wild-type levels of luminescence and luciferase in the cya-like mutant but not in the crp-like mutant. The results are consistent with the hypothesis that in V. fischeri cyclic AMP and cyclic AMP receptor protein are required for induction of the luminescence system.
TL;DR: It is thought that a similarity may exist between the non-classical 5-HT receptor, coupled with an adenylate cyclase in colliculi neurons, and the non'-HT receptor', involved in gastric and ileum motility which is specifically stimulated by substituted benzamide derivatives in the same order of potency (metoclopramide, BRL 24924).
TL;DR: Results indicate that beta gamma subunits influence phospholipase C activity in a concentration range similar to that necessary for regulation of adenylate cyclase activity and suggest the involvement of a G-protein possessing an alpha beta gamma heterotrimeric structure in coupling hormone receptors to phospholIPase C.
TL;DR: An effect of 17β‐estradiol on DA target cells in both systems is demonstrated, suggesting that the steroid modifies the coupling process by a mechanism that still has to be elucidated.
Abstract: Primary cultures of anterior pituitary cells from female rats and of mouse embryonic striatal neurons were used to study the effects of 17 beta-estradiol on D1- and D2-dopamine (DA)-sensitive adenylate cyclase. 17 beta-Estradiol pretreatment (10(-9) M, 72 h) suppressed the D2-DA-induced inhibition of adenylate cyclase activity in anterior pituitary cells. The steroid (10(-9) M, 24 h) also blocked the D2-DA-evoked response in striatal neurons whereas it enhanced by twofold the D1-DA-induced stimulation of the enzyme activity in these neurons. All these effects of the steroid were dose dependent and specific, as neither 17 alpha-estradiol, dexamethasone, nor progesterone used at the same concentration (10(-9) M) was effective. Furthermore, the modulation of DA-sensitive adenylate cyclases by the steroid required long-term exposure of living cells to 17 beta-estradiol since neither 17 beta-estradiol pretreatment for 4 h nor its addition to broken cells directly into the adenylate cyclase assay induced any alteration in the DA-sensitive adenylate cyclase activity. These results are in agreement with a genomic effect of the steroid. Using both anterior pituitary cells and striatal neurons in culture, 17 beta-estradiol affected neither the total number of DA (D1 and D2) receptors nor the estimated number of adenylate cyclase catalytic units. Therefore, it is suggested that the steroid modifies the coupling process by a mechanism that still has to be elucidated. These results demonstrate an effect of 17 beta-estradiol on DA target cells in both systems.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The present results provide an example of positive interaction between cAMP and the phosphoinositide signaling system and suggest that a coexisting neuropeptide may exert pleiotropic actions upon its target cell by stimulating multiple signal transduction pathways.